1. Field of the Invention
THIS INVENTION relates to immunostaining and especially immunocytochemical (ICC) staining of tissue specimens for diagnostic and related purposes.
2. Background Discussion and the Prior Art
The immunostaining technique, while increasingly widely practiced and subject to the development of automated procedures, is nevertheless difficult to control and critically dependent, for reproducible results, on rigid adherence to operating procedures and conditions and, especially, reagent quality and consistency. Even then the experience of the operator is vital to interpretation of results. Thus the assessment and interpretation of an ICC stain requires considerable experience to discriminate between what is desired specific staining of the chosen antigen and what is artefactual staining of undesirable tissue substrates.
Many parameters are involved in the overall procedure by which a stained specimen is obtained from an original tissue sample and some of those parameters are out of the control of the operator. Those within his control and which may be optimised to preserve and maximally demonstrate the chosen antigen include: Fixation - type of fixative and duration of fixation Processing - types of reagents and duration of contact with each
______________________________________ ICC staining - method; reagent concentrations; incubation times; visualising agent ______________________________________
Within each category there are further subdivisions that require control and standardisation. Considering the last category, ICC staining, the operator is not only dependant upon reagent quality but also upon method reproducibility. He therefore assumes that his reagent supplier can guarantee the quality and consistency of reagents so that by eliminating this variable he can direct his efforts to reproducing and standardising his method consistently. This is practicable because variations in the quality of reagents generally only appear in comparisons of the products of different suppliers, accordingly it is quite common for ICC method operators to use reagents from one particular supplier only.
Individuals performing ICC methods, however, do vary in their technique and ability, and whilst method protocols are usually strictly laid down, results are seldom identical between any two operators performing the same method.
A certain degree of control is introduced by using "known positives" and seldom is any interpretation made independently of this control. However, the importance of the preparative, e.g. fixation and processing, steps prior to ICC staining cannot be fully realised, because it has to be assumed that the control or "test" tissue subjected only to the staining routines has previously been prepared by more or less the same protocols as the tissue sample under investigation. Unfortunately this cannot always be the case, especially if material is received from other sources; for instance, typical tissue fixation and processing procedures can account for anything from 30-100% loss of antigenicity from the material. The ICC operator is therefore presented with insurmountable problems when achieving weakly positive or unexpectedly negative results and must try to establish the reason and rectify it by adjusting one of his methods accordingly.
Many of these problems would be overcome if a quantitative "control" could be used at all stages of preparation and staining to evaluate the contribution that each one makes to the overall ICC result.
This control should also take the subjectivity of assessment out of ICC interpretation and indicate the advantages of the use of certain reagents over others in preserving and demonstrating the maximum amount of antigen in tissues.
Accordingly we have been investigating the possibilities for providing a test material that can be subjected to all the preparative procedures to which a tissue sample is subjected and that can therefore be "prepared" alongside a tissue sample to provide a proper basis of comparison with the results of corresponding preparation of the tissue sample.
It was necessary first to demonstrate the practicality of providing on a support, such as to be provided by the sought test material, a range of identifiable standardised specific antigen concentrations capable of being usefully processed by recognised ICC routines to yield consistent and reproducible results, perhaps after extended storage periods prior to processing.
To this end, and based on a study by Scopsi and Larsson (Histochemistry 1986, 84:221) into the increased sensitivity in peroxidase immunocytochemistry, an improved, simplified test material model was prepared for such an investigation. Nitrocellulose filters of 0.2 .mu.m pore size were spotted with 2 .mu.l droplets of different dilutions of human serum representing different specific antigen concentrations. The serum adsorbed formed spots of 2-3 mm diameter which were then dried, fixed and stained with Sternberger's PAP procedure. Various routines were tested.
It was found that such preparations, once fixed, are stable for many months, yielding reproducible results to a consistent end-point. Other findings include:
1. The existence of optimum dilutions for immune reagents.
2. Reduction in background with a non-ionic detergent such as Triton X in the buffer wash system.
3. Reduction in background with agitation of the buffer wash.
4. Visualisation with DAB chromagen is more sensitive than with AEC.
5. Addition of imidazole to DAB chromagen further improves visualisation.
6. Overnight incubation enables the use of more diluted primary antisera but leads to a slight increase in background.
7. Effect and duration of heat of fixation significantly reduces staining intensity.
8. Slight antigen loss occurs with aqueous formalin fixation.
Unfortunately the model, in this form, cannot be used to look at the effects of tissue processing routines, because nitrocellulose is soluble in alcohols and cannot, therefore, be subjected to such routines.
Paper filters of various grades and makes were similarly assessed in an attempt to overcome the problem of solvency in alcohol. These were found to be unsuitable owing to the following factors:
1. The area of diffusion was too large.
2. Fibres were shed, especially during agitated washing stages, leading to eventual breakdown of the filter paper.
3. Filters became saturated after the addition of two drops of immunoreagent during ICC staining.
4. The final end-point was difficult to determine due to the diffuse area of positivity.
5. All filters demonstrated intense non-specific background staining.
An attempt was made to use cellulose acetate in the same way, but again the spots became diffuse and although this material is resistant to tissue processing alcohols and xylene, considerable fading of the preparations was noticed upon drying out at the end of the procedure.
These findings led us to develop a second model, which used agar as a support medium for the dilutions of serum: as a 1.5% gelled solution, the following advantages are offered:
1. Agar is insoluble in processing reagents
2. It will not melt below 85.degree. C., and is therefore compatible with molten wax (m.p. 60.degree. C.)
3. It allows the diffusion of antibodies and antigens
4. It only stains with acidic dyes
5. It is easy to section
6. It is cheap and widely available.
Moulds of 2.5 cm diameter and 0.5 cm depth were made to receive molten agar. There were 12 circular pegs in each mould so that the agar disc prepared would contain 12 wells. This was to enable small volumes of serum dilutions to be held in defined locations of the disc until diffusion had taken place.
In a series of tests, following diffusion of the serum dilutions into the disc from the well sites, the discs were aqueously fixed and then processed routinely by an overnight schedule using an automatic tissue processing machine such as disclosed in EP-0,077,477.
Sections 5 .mu.m thick obtained by microtomy of the processed discs were stained by the PAP method. Serum as an antigen source processed in this way showed positive staining by ICC, thus proving the feasibility of the technique. Problems became apparent, however, due to the different rates of diffusion shown by the different serum concentrations. As dilutions had been made in Tris-buffered saline (TBS), the higher serum concentrations showed a tendency to diffuse further into the agar. It also became apparent that rates of diffusion were not constant and depended upon the temperature at which the processing occurred.
Another factor influencing the diffusion was found to be time, since even after diffusion appeared to be complete, a dye model showed it to be a continuing process, with the boundaries of the agar disc being the limiting factor. Attempts to overcome these problems were made by:
(i) using solutions of similar viscosity by making the dilutions of human serum in different inert animal sera PA1 (ii) setting a limit to diffusion times prior to fixation.
Animal sera, generally, gave decreased sensitivity after ICC staining. This could be due to the cross-linking of serum antigens and animal serum protein by formaldehyde molecules.
Other minor problems with agar were encountered in sectioning and drying. Preparations appeared to shrink slightly whilst on the water bath. Picking up sections onto glass slides (coated with poly-L-lysine adhesive) and drying was not always successful as occasional floating off of sections was encountered at some stage during the process.